Fungal glucoamylases

Fungal glucoamylases Dariush Norouzian b,*, Azim Akbarzadeh a, Jeno M. Scharer b, Murray Moo Young b b

a Pilot Biotechnology Department, Pasteur Institute of Iran ,Tehran 13164, Iran Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1

Received 1 February 2005; accepted 22 June 2005 Available online 8 August 2005

Abstract Fungi are employed to produce industrially important glucoamylases. Most glucoamylases are glycosylated. Glycosylation enhances the enzyme stability. Glucoamylases contain both starch binding and catalytic binding domains, the former being responsible for activity on raw (insoluble) starch. Proteases may act on this domain causing the enzyme to lose its activity on insoluble starch. Optimal activity is observed at pH 4.5 to 6.5 and 50 to 70 8C. Glucoamylases contain up to 7 sub-sites with highly varying affinity. They can be produced by different methods including submerged, solid state and semi-solid state fermentation processes. D 2005 Elsevier Inc. All rights reserved. Keywords: Glucoamylase function; Fungal fermentation; Fungal enzymes; Biochemical properties; Kinetic characteristics; Industrial production

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . Fungi-producing GA . . . . . . . . . . . . . . . Enzyme production . . . . . . . . . . . . . . . . Enzyme characteristics . . . . . . . . . . . . . . 4.1. Molecular weight and structure . . . . . . 5. Kinetic characteristics . . . . . . . . . . . . . . . 5.1. Optimal conditions for catalytic activity . . 5.2. Subsite binding affinities of glucoamylases 6. Conclusions . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction * Corresponding author. Present address: Pilot Biotechnological Department, Pasteur Institute of Iran, Tehran 13164, Iran. E-mail address: [email protected] (D. Norouzian).

Glucoamylase (GA), also known as amyloglucosidase or g-amylase (EC 3.2.1.3), is a biocatalyst capable of hydrolyzing a-1,4 glycosidic linkages in raw (sparse-

ly soluble) or soluble starches and related oligosaccharides with the inversion of the anomeric configuration to produce h-glucose. In addition to acting on a-1,4 linkages, the enzyme slowly hydrolyzes a-1,6 glycosidic linkages of starch (Weil et al., 1954; Pazur and Ando, 1960; Koshland, 1953; Fierobe et al., 1998). The specific activity (K cat / K m) towards the a-1,6 linkage is only 0.2% of that for the a-1,4 linkage (Hiromi et al., 1966; Sierks and Svensson, 1994; Frandsen et al., 1995; Fierobe et al., 1996). This adversely affects the yield in industrial applications of saccharification (Sauer et al., 2000). An increase of the glucose yield in saccharification beyond the present 96% level can be achieved by suppressing the activity of GA on the a-1,6 linkages present in starchy materials (Sauer et al., 2000). The widely accepted mechanism of hydrolysis involves proton transfer from the catalyst to the glycosidic oxygen of the scissile bond. A general acid–base catalyst (McCarter and Withers, 1994; Sinnot, 1990; Konstantinidis and Sinnot, 1991; Tanaka et al., 1994) donates hydrogen to the glucosidic oxygen and a catalytic base guiding the nucleophilic attack by a water molecule on the C-1 carbon of the glucose moiety. The amino acid residue, Glu 179 of glucoamylase produced by Aspergillus niger has been identified as the general acid catalyst, and Glu 400 as the probable catalytic base group (Harris et al., 1993; Sierks et al., 1990; Frandsen et al., 1994). Glucoamylases are industrially important biocatalysts and have extensive uses in the manufacture of crystalline glucose or glucose syrup either as soluble or immobilized enzymes (Abraham et al., 2004; Torres et al., 2004; D’Souza and Kubal, 2002; Ruadze et al., 2001). Efforts are being made to produce and study the characteristics, structure, and function of this valuable biocatalyst from different primarily fungal sources. 2. Fungi-producing GA Many fungal species are capable of producing GA under different fermentation conditions and techniques. Most attempts involved seeking fungal species capable of hydrolyzing raw starch at elevated temperatures. The various fungi synthesizing GA that is active at higher temperatures include Aspergillus awamori, Aspergillus foetidus, Aspergillus niger, Aspergillus oryzae, Aspergillus terreus, Mucor rouxians, Mucor javanicus, Neurospora crassa, Rhizopus delmar, Rhizopus oryzae (Pandy et al., 2000) and Arthrobotrys amerospora (Jaffar et al., 1993; Norouzian and Jaffar, 1993). However, the industrial focus has been on GA from Aspergillus

niger and Rhizopus oryzae. The employment of GAs from these sources in the starch processing industries is due to their good thermostability and high activity at near neutral pH values (Frandsen et al., 1999; Reilly, 1999). 3. Enzyme production Glucoamylases can be produced by submerged, solid state and semi-solid state fermentation using stirred tank vessels, airlift reactors or stacked trays. GA production is influenced by bioreactor design and operating mode. Bioreactors that had been employed to study GA production included flasks, trays, rotary reactors, columns (both horizontal and vertical) bioreactors (Pandy and Radhakrishna, 1992). Bo et al. (1999) investigated the production of glucoamylase in an air-lift bioreactor employing Rhizopus oligosporus. Glucoamylase was produced by a recombinant Aspergillus niger in glucose limited chemostat supplemented with various organic nitrogen sources (Richard et al., 2000). It was observed that cultures supplemented with various organic nitrogen sources including L-alanine, L-methionine, casamino acids, or peptone had shown reduced specific glucoamylase production. Fujio and Morita (1996) and Ramadas et al. (1996) employed Rhizopus SP.A-1 and Aspergillus niger to produce GA by solid state and semi-solid state fermentation. They found higher GA titer in the semisolid fermentation system. Pedersen et al. (2000) compared GA production in batch, continuous and fed batch operations and found maximum production during batch cultivation. 4. Enzyme characteristics Glucoamylases of fungal origin usually occur in multiple forms (Manjunath et al., 1983; Miah and Ueda, 1977; Pazure et al., 1971) and these multiplicities may be related to either the activity of protease produced along with GA concomitantly or, as pointed out by Svensson et al. (1986), the forms may be derived by different secondary processing. Fungal glucoamylases have two domains, namely a catalytic domain and a starch binding domain. The two domains are connected by an O-glycosylated polypeptide linker located at the N-terminus. The starch binding domain of GA plays an active role in hydrolyzing raw starch and supports the enzyme adsorption to the cell wall where local increase of enzyme concentration may result in enhanced glucose flow to the cell (Kaneko et al., 1996; Neustroev et al., 1993). Partial or total

proteolytic excision of the starch binding domains leads to the formation of GA capable of degrading soluble starch only (Cutinho and Reilly, 1997). Therefore, an important objective to enhance GA activity toward raw starch is to reduce protease production during GA fermentation. To accomplish this, the operational approaches that have been successful include: a) cell immobilization (Liu et al., 1998); b) growth morphology (Gregg et al., 2001); c) pH control (Bertolin et al., 2003) ; d) nutrient control (Pedersen et al., 2000) ; e) bioreactor configuration (Pandy and Radhakrishna, 1992); and f) the use of protease inhibitors (Xu et al., 2000). 4.1. Molecular weight and structure The molecular weight of GAs from various fungal sources is usually in the range of 48 to 90 kD excepting a few 125 kD GAs produced by Aspergillus niger (Suresh et al., 1999). The carbohydrate content of fungal GA is reported to be usually in the range of 10–20% of the molecular mass (Pazure et al., 1971; Ueda, 1981). For example, glucoamylase C and D from Rhizopus niveus contain 14.9% and 12.7% carbohydrate, respectively (Ueda, 1981), while GA produced by Neurospora crassa is reported to have only 5.1% carbohydrate (Spinelli et al., 1996). The polysaccharide residue does not seem to affect the protein’s tertiary structure, but the elimination of glycosylation in recombinant Aspergillus awamori led to a decrease in enzyme secretion and thermal stability. Three dimensional structural studies of glycosylation in GA of Aspergillus awamori X-100 suggested that without glycosylation the protein could make steric contact with the linker thus negatively affecting the stability of the enzyme (Cutinho and Reilly, 1997). Generally O-glycosylation has been found to link the carbohydrate moiety to N-terminus in Aspergillus niger. Low density glycosylation around the catalytic domain was reported to cause rigidity of the catalytic domain. Further glycosylation rigidifies the long amino acid residue linker in solvated environments as well thus contributing to the physical separation of catalytic domain and starch binding domains. Studies indicate that the carbohydrate moiety, in addition to a stabilizing effect, also prevents the unfolded and partially folded proteins to aggregate. Yet another role of Oglycosylation in GA is to protect the enzyme from proteolysis. It has been found that GAs with lower degree of glycosylation are more prone to protease action (Cutinho and Reilly, 1997; Le Gal-Go¨efer et al., 1995).

5. Kinetic characteristics 5.1. Optimal conditions for catalytic activity Fungal glucoamylases are usually most active at acidic pH values, but the various forms have different pH optima. For example one of the three GAs produced by a nematophagus fungus, Arthrobotrys amerospora was active at pH 6.0 and the other two at pH 5.6 (Norouzian et al., 2000). Commercial preparation of glucoamylase from Aspergillus niger consisted of at least six forms with pH optima ranging from 3.5 to 5.0 (Ono et al., 1988). Spinelli et al. (1996) reported that GA obtained from a mutant strain of Neurospora crassa is optimally active at pH 5.4. Glucoamylases produced by Monascus kaofieng nov-sp. F-1 had two active forms with optimal pH of 4.5 and 4.7, respectively. The stability of fungal glucoamylases is also pH dependent. For example, the GAs produced by Aspergillus terreus and Aspergillus terreus NA-170 were stable over the pH range of 3 to 7 (Ali et al., 1991; Ghosh et al., 1991). Many glucoamylases function at thermophilic temperatures, usually 50 to 60 8C. The enzymes from Aspergillus niger NRRL 330 and Aspergillus awamori var. kawachi were optimally active at 50 8C and 60 8C, respectively whereas GAs of Arthrobotrys amerospora were optimally active at 55 8C (Venkatatramu et al., 1975; Spinelli et al., 1996; Norouzian et al., 2000). In a few instances, higher optimum temperature had been reported. For example, the optimum temperature for glucoamylase activity of the filamentous fungi Trichoderma reesi and Scytalidium thermophilium was reported to be 70 8C (Fagerstom and Kalkkinen, 1995; Aquino et al., 2001). Many fungal glucoamylases have been reported to be stable at considerably higher temperatures for short time periods (Fagerstom and Kalkkinen, 1995; Okolo et al., 2001). 5.2. Subsite binding affinities of glucoamylases According to Hiromi et al. (1973, 1983) the cleavage of various a-glucosidic bonds occurs at the same active site in the enzyme molecule. Therefore, the substrate can bind to GAs to form two different ends, either a productive or non-productive end. In the former, oligosaccharide will occupy at least the first and second subsite; hence, the susceptible glycosidic linkages will be accessible for catalysis. In GA the catalytic site is located between sub-site no.1 and 2 (note that GA acts in exo mode), therefore, there is only one productive end. Also any substrate binding which does not

lead to the hydrolysis can be termed as non productive. This is illustrated in the diagram below:

K

nP

ESnp

Kint

E+P

E + Sn

The enzyme cleaves a-1,4 glycosidic linkages preferentially, but a-1,6 linkages are also attacked, albeit at a slower rate. The low specific rate of a-1,6 cleavage adversely affects enzyme kinetics and saccharification efficiency. Glucoamylases from various sources are reported to have optimum specific activities over a wide temperature and pH range. For industrial applications, optimum enzyme activity at 50–60 8C and at near neutral pH is preferred. References

K

nq

ESnq

When a linear polymeric substrate (Sn) with degree bnQ polymerization (DP) binds to the enzyme molecule, it can form one or more productive (ESnp) and several non-productive (ESnq) complexes. The productive and non-productive complexes have different substrate affinity constants (K n,p and K n,q). Based on kinetic studies of GAs produced by Aspergillus saitoti, Aspergillus oryzae, Aspergillus niger and Arthrobotrys amerospora, it appears that glucoamylases have seven subsites for substrate binding (Hiromi et al., 1973; Takashi et al., 1984; Meagher et al., 1989; Kazuhisa et al., 1988; Christensen et al., 1997; Norouzian et al., 2000). Glycosidic linkages of maltose oligomers are cleaved between sub-site 1 and 2. The other sub-sites possess variable affinities for the substrate. The affinity of the first sub-site is very low. In contrast, sub-site 2 has the highest affinity and the affinity of the individual sites decreases from sub-site 3 to 7. 6. Conclusions The starch processing industry employs glucoamylases mainly from Aspergillus and Rhizopus species carry out starch saccharification. Although fungal glucoamylases may be produced by several fermentation methods, a semi-solid state batch fermentation system is reported to give the highest enzyme titer. Glucoamylases have separate substrate binding and catalytic domains. A major operational problem is the proteolytic inactivation of the substrate binding domain rendering the enzyme incapable to attack raw starch. Undesirable protease production during fermentation can be alleviated by cell immobilization, nutrient and pH control, bioreactor design, and use of protease inhibitors. The catalytic domain consists of several sub-sites with varying affinity for cleaving glycosidic bonds.

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